Kinetics of Milk Lipoprotein Lipase.. Studies with Tributyrin

Abstract

Tributyrin offers several advantages as a substrate for lipases. It is readily dispersed without detergents and the products formed on hydrolysis are water-soluble. However, in this system lipoprotein lipase is not stable and the rate of hydrolysis decreases rapidly. Inactivation is promoted by an increase of the pH or the concentration of sodium chloride, but is impeded by surface-active materials such as proteins and detergents. Because of the pronounced effects that these components have on the stability of the enzyme it has been difficult to evaluate their effects on the catalytic properties in the tributyrin system. These difficulties could be largely overcome by using a gumarabic-stabilized emulsion of tributyrin and initiating the reaction at pH below 8. Under these conditions there was a short initial phase during which the rate of lipolysis increased by the reaction then continued at a steady rate, suggesting that the enzyme was stable at the emulsion-water interface. By adding substances or by changing the pH after lipolysis had attained a steady rate, it was possible to evaluate the effects on the rate of hydrolysis under conditions when the enzyme was rather stable. Using this approach it was found that the enzyme hydrolyzes tributyrin at about the same rate as it hydrolyzes long-chain triglycerides. The rate of hydrolysis increased with pH to 9.5; higher pH could not be adequately tested because of rapid non-enzymatic hydrolysis. There was no need for cofactor protein or albumin; agents which are necessary for sustained rapid hydrolysis of long-chain triglycerides. Larger amounts of albumin or of several other proteins inhibited the lipolysis, presumably since the proteins adsorb to the emulsion-water interface. This inhibition could be partly relieved by cofactor proteins. This was the only case when an effect of such proteins was seen in the tributyrin system. Detergents such as bile salts and decyl sulphate inhibited lipolysis. At pH 7.8 90 inhibition was obtained with 0.5 mM deoxycholate, but the inhibition was less marked at higher pH. It was not relieved by pancreatic colipase or by any of several proteins with cofactor activity for lipoprotein lipase. Many of the agents reported to strongly affect lipoprotein lipase activity against long-chain triglycerides in the presence of albumin and cofactor proteins had little or no effect in the tributyrin system, e.g. 1 M NaC1, 5 mM pyrophosphate, 5 mM Ca2+, or heparin or protamin in concentrations up to 0.1 mg/ml.

Interactions of lipoprotein lipase with the active‐site inhibitor tetrahydrolipstatin (Orlistat)R

“…This is in accord with the effects of apo CII on the catalytic activity of LPL. With tributyrylglycerol apo CII has little or no effect (Rapp and Olivecrona, 1978), while experiments with the emulsion of trioleoylglycerol under the conditions used here showed that apo CII stimulated the activity about threefold. It should be realized that the values for K, and k,, can not be directly used for characterization of the interaction between LPL and THL, because they are also dependent on yet unknown values for the interfacial concentration of the substrate molecules and their binding constants to the enzyme.…”

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

“…Thus, apoCII is not needed for interfacial binding of LPL. ApoCII has effects on LPL activity only with triacylglycerols and phospholipids that contain acyl chains longer than seven carbons (23)(24)(25). The level to which apoCII activates LPL in vitro depends not only on the substrate molecules but also on components like surfactants and other proteins that are present in the system (21).…”

Chymotryptic cleavage of lipoprotein lipase

“…The activity against tributyryl glycerol was measured according to the method described by Rapp and Olivecrona (1978). Substrate emulsion was freshly prepared by sonication of tributyrin (0.5 ml) in a 16% (masdvol.)…”